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The SUMO ligase MMS21 profoundly influences maize development through its impact on genome activity and stability

['Junya Zhang', 'Department Of Biology', 'Washington University In St', 'Louis', 'St. Louis', 'Missouri', 'United States Of America', 'Robert C. Augustine', 'Masaharu Suzuki', 'Department Of Horticultural Sciences']

Date: 2022-01 

The post-translational addition of SUMO plays essential roles in numerous eukaryotic processes including cell division, transcription, chromatin organization, DNA repair, and stress defense through its selective conjugation to numerous targets. One prominent plant SUMO ligase is METHYL METHANESULFONATE-SENSITIVE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, which has been connected genetically to development and endoreduplication. Here, we describe the potential functions of MMS21 through a collection of UniformMu and CRISPR/Cas9 mutants in maize (Zea mays) that display either seed lethality or substantially compromised pollen germination and seed/vegetative development. RNA-seq analyses of leaves, embryos, and endosperm from mms21 plants revealed a substantial dysregulation of the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves and altered accumulation of mRNAs associated with DNA repair and chromatin dynamics. Interaction studies demonstrated that MMS21 associates in the nucleus with the NSE4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex, with in vitro assays confirming that MMS21 will SUMOylate SMC5. Comet assays measuring genome integrity, sensitivity to DNA-damaging agents, and protein versus mRNA abundance comparisons implicated MMS21 in chromatin stability and transcriptional controls on proteome balance. Taken together, we propose that MMS21-directed SUMOylation of the SMC5/6 complex and other targets enables proper gene expression by influencing chromatin structure.

The post-translational addition of SUMO to other proteins by the MMS21 SUMO ligase has been implicated in a plethora of biological processes in plants but the identit(ies) of its targets and the biological consequences of their modification remain poorly resolved. Here, we address this issue by characterizing a collection of maize mms21 mutants using genetic, biochemical, transcriptomic and proteomic approaches. Our results revealed that mms21 mutations substantially compromise pollen germination and seed/vegetative development, dysregulate the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves, increase DNA damage, and alter the proteome/transcriptome balance. Interaction studies showed that MMS21 associates in the nucleus with the NON-SMC-ELEMENT (NSE)-4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex responsible for DNA-damage repair and chromatin accessibility. Our data demonstrate that MMS21 is crucial for plant development likely through its maintenance of DNA repair, balanced transcription, and genome stability.

Funding: This work was supported by grants from the NSF-Plant Genome Research Program to RDV (IOS-1546862) and to DRM and MS (IOS-1748105) https://nsf.gov/funding/pgm_summ.jsp?pims_id=5338 , and a NIH NRSA Ruth L. Kirschstein postdoctoral fellowship (5 F32 GM103161) to RCA https://researchtraining.nih.gov/programs/fellowships/f32 . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we further investigated the functions of MMS21 in maize (Zea mays) using a library of UniformMu transposon-insertion and CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9-induced mutations. These analyses revealed that MMS21 is essential in maize with critical roles in root, shoot, pollen, and seed development. While the exact mechanism(s) are not yet clear, defects in DNA repair, and mis-regulated proteome/transcriptome balance were evident for the mms21 germplasm. Interaction studies, RNA-seq analyses, and in vitro SUMOylation assays collectively linked MMS21 to the NSE4 and SMC5 subunits of the SMC5/6 complex, suggesting that MMS21-directed SUMOylation of the SMC5/6 complex and possibly other targets are essential for proper chromatin function and subsequent maize development.

MMS21, in particular, has garnered interest given its high conservation among eukaryotes and its potential action in a variety of nuclear processes. Arabidopsis mms21 null mutants are viable but develop stunted roots, altered apical meristems, dwarfed rosettes, and higher chromosome ploidy numbers in both somatic tissue and male gametes, suggestive of roles in the cell cycle and endoreduplication [ 12 , 28 , 29 ], while the null mms21 mutants in rice also have stunted vegetative growth [ 19 ]. More recently, MMS21 was linked to the DNA damage response and chromatin structure potentially through the respective modification of BRAHMA, a conserved ATPase component within the SWI/SNF chromatin-remodeling complex, and the cell-cycle check point protein DPa, [ 18 , 30 , 31 ]. In other organisms, MMS21 has been shown to be a crucial component of the nuclear STRUCTURAL MAINTENANCE OF CHROMOSOMES-5/6 (SMC5/6) complex, which is an evolutionarily-conserved ATPase that influences chromatin compaction and is required for recombinational DNA repair, replication fork restart, ribosomal DNA and telomere maintenance, and genome stability [ 32 ]. In accord, MMS21 has been found associated with Arabidopsis SMC5/6 complex [ 13 ], with one possible target being the integral NSE4 subunit [ 5 ].

To date, three classes of SUMO E3s have been characterized in plants: SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZ)-1, PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIAL)-1/2, and METHYL METHANESULFONATE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, referred to here as MMS21 [ 1 , 2 ]. They share a SP-RING domain that binds the E2-SUMO intermediate [ 26 ], along with a variety of other motifs that presumably identify specific substrates and/or help anchor the E3 to appropriate surfaces/complexes, including DNA and methylated histones. While SUMOylation by the SIZ1 E3 has been connected to a wide range of cellular events and substrates in plants, especially those related to stress defense [ 5 , 14 , 21 , 23 , 27 ], the function(s) of the other E3s are currently unclear.

SUMOylation is driven by an ATP-dependent enzymatic cascade involving the sequential action of a SUMO-activating enzyme (or E1) and a SUMO-conjugating enzyme (or E2) that prepares SUMO for addition, and in most cases, a SUMO-protein ligase (E3) that identifies appropriate substrates and encourages transfer from a thioester-linked E2-SUMO donor [ 1 , 2 ]. While some substrates become modified with a single SUMO, others become iteratively modified with multiple SUMOs attached either at multiple lysines within the substrate or to previously bound SUMOs connected internally through SUMO-SUMO isopeptide linkages. The conjugated SUMOs can also be ubiquitylated by a family of SUMO-targeted ubiquitin ligases, thus merging the influence of these two ligation systems. SUMO attachment is often reversible through a collection of deSUMOylating enzymes that specifically cleave the isopeptide bond between the SUMO moiety and target lysines [ 25 ].

Over the past decade, in-depth proteomics have identified over a thousand SUMO substrates in the eudicot Arabidopsis thaliana [ 3 – 6 ], with companion genetic studies providing links between SUMOylation and a wide array of cellular processes. Included are regulations of gamete formation and embryogenesis, leaf development, root stem cell maintenance, hormone signaling, light perception, circadian rhythm entrainment, phosphate acquisition, transcriptional and epigenetic regulation, DNA damage repair, and defense against various abiotic and biotic challenges [ 2 , 7 – 19 ]. Particularly notable is the rapid SUMOylation of numerous proteins when plants are subjected to pathogen attack and heat, drought or salt stress, which presumably provides protection by yet to be fully understood mechanism(s) [ 14 , 20 – 24 ]. Considering that SUMOylation regulates physiological and developmental processes crucial to agriculture, uncovering the molecular mechanisms underpinning selective SUMOylation might reveal novel strategies for crop improvement, especially in suboptimal environments.

Plants like other cellular organisms exploit a plethora of post-translational modifications to expand the functionality of their proteomes, including controls on enzymatic activity, subcellular location, interaction with other effectors, and ultimately on the turnover rates of the affected proteins. One reversible modification that is emerging as a key regulator involves attachment of the ~100-amino-acid protein SMALL UBIQUITIN-LIKE MODIFIER (SUMO), which is structurally related to ubiquitin and likewise becomes covalently linked via an isopeptide bond to accessible lysines within its targets [ 1 , 2 ].

Results

mms21 mutants have relatively normal SUMOylome profiles As one strategy toward understanding how MMS21 affects maize, we assessed its overall impact on SUMOylation by subjecting total tissue extracts to immunoblot analysis with anti-SUMO antibodies [24]. As shown in Fig 4A, SUMO-conjugate profiles in mms21-1 and mms21-2 leaves were mostly indistinguishable from those seen in W22 both before and after a 30-min heat stress at 42°C, which dramatically increases the pool of SUMO conjugates [24]. No species were absent in mms21-1/2 leaves with or without the heat stress, and at most, only a few new species at ~60 and 37 kDa appeared. Similarly, we tested embryo and endosperm tissue harvested from seeds at 16 DAP (Fig 4B). Again, little differences in the profiles and levels of SUMO conjugates and free SUMO were detected in the mutants, strongly suggesting that MMS21 modifies only a small subset of SUMO substrates in maize, consistent with similar studies with Arabidopsis [5]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Profiles of SUMO and SUMO Conjugates are Weakly Altered in mms21 Mutants. Total protein was extracted from the indicated tissues and subjected to SDS-PAGE and immunoblot analysis with anti-SUMO1 antibodies. Near equal protein loading was confirmed by immunoblotting with anti-histone H3 antibodies. Closed arrowheads and brackets locate free SUMO and SUMO conjugates, respectively. Open arrowheads locate proteins that differentially accumulated in the mms21 backgrounds. Asterisks locate unidentified species that react with the anti-SUMO1 antibodies. (A) SUMO conjugates accumulating in seedling leaves before (-) or after (+) a 30-min heat shock at 42°C. (B) SUMO conjugate profiles in embryo and endosperm tissues collected at 16 DAP. https://doi.org/10.1371/journal.pgen.1009830.g004

mms21 mutations alter the transcriptome/proteome balance To further assay the impact of MMS21 on protein accumulation, we compared the proteomes of mms21 mutant and normal siblings by shot-gun mass spectrometry (MS) of young seedlings [42]. Here, trypsinized total protein extracts from 10-DAS seedlings were subjected to reversed-phase separation followed by tandem MS, which allowed relative quantification for approximately 4,000 proteins from the MS1 scans based on the mean of three biological replicates each with two technical replicates. The proteome data were then normalized among samples using a list of 150 proteins relatively unaffected by the mutations [42], which was then validated by assaying the levels of abundantly detected histones which should be consistent across genotypes (Figs 7A, S9A and S9B). PPT PowerPoint slide

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TIFF original image Download: Fig 7. mms21 Mutants have Altered Proteome Profiles and Reduced SMC5 Levels. (A) Altered proteome profile for the mms21-2 mutant. The volcano plot depicts protein abundance changes for 4,084 proteins detected from mms21-2 leaves as compared to those of its normal sibling. Each dot represents one protein that had detectable expression in both samples and was plotted based on its log 2 FC in abundance (mutant/normal siblings) and its -log 10 p-value of significance based on the three biological replicates, each with two technical replicates. The horizontal and vertical dashed lines mark a FC = 2 in protein abundance and a p-value = 0.05, respectively. Histone proteins used to confirm data normalization are shown as green. SUMO pathway components and DNA repair-associated proteins are highlight in blue and red respectively. (B) GO analysis of significantly regulated proteins in the mms21 mutant versus W22 seedlings. The vertical coordinates indicate the enriched GO terms, and the horizontal coordinates show the number of genes for each GO term when comparing differentially expressed genes common between mms21-1 and mms21-2 seedlings. Negative values indicate downregulation, positive values indicate upregulation. GO enrichment was performed using all the three sub-ontologies: ‘biological process’, ‘molecular function’, and ‘cellular component’. (C) SMC5 protein abundance is reduced in strong mms21 mutant backgrounds. Relative protein abundances of SMC5 from the mms21-1, mms21-2 and mms21-CR1 mutants and their normal siblings. Each bar represents the mean of three biological replicates(±SD). (D) Positive correlation between transcriptome and proteome data in mms21 mutants. Scatter plot showing the relationship between changes in protein and mRNA abundances for the mms21-2 mutant versus normal sibling as determined by plotting the log 2 FC in mRNA abundance versus the log 2 FC in protein abundance. Red dots (154) and blue dots (277) highlight proteins that were more or less abundant (FC>2), respectively, in both mms21-1 and mms21-2 leaves as compared to their normal siblings. The black and dashed blue lines show the correlations for mms21-2 and for the combined data of mms21-1 and mms21-2 (red and blue dots). https://doi.org/10.1371/journal.pgen.1009830.g007 When the normalized proteome data were displayed by volcano plots that assessed both log 2 FC in abundance versus p-values of significance, numerous proteins were significantly more or less abundant in the mms21 seedlings compared to their normal siblings based on a FC = 2 threshold and adjusted p-values <0.05. For the mms21-2 background, the values were 476 up- and 569 downregulated (26% mis-accumulated), while for the mms21-1 background, the values were 496 up and 640 downregulated (27% mis-accumulated) as compared to 9% of the proteins having different values (>2 FC, p-value <0.05) when comparing normal siblings of mms21-2 and mms21-1 to each other (Figs 7A, S9A and S9B). (We presume that most of the proteins assigned as differentially accumulating between the normal siblings represent noise inherent to MS data collection and analysis.) As with the immunoblot assays, we failed to detect any zein storage proteins by MS in the mms21 seedlings despite having elevated mRNAs. When the differentially accumulating list was subjected to GO analysis, a broad spectrum of protein functionalities was impacted in the mms21 backgrounds, such as ‘cellular process’, ‘metabolic process’ and ‘catalytic activity’, with little selective impact seen on specific subcategories, thus likely reflecting a general alteration in protein composition (Fig 7B). We then compared the differences in protein abundance for the mms21-2 seedlings versus normal siblings with those described above for the corresponding transcripts for 3,756 proteins with data available for both. Surprisingly, a modest but significant correlation was seen (R2 = 0.42), with the proteins less abundant in the mutant also having less mRNA, while those proteins more abundant in the mutant also having more mRNA (Fig 7D). This correlation was even more robust (0.69) when we analyzed only those proteins (431 total) that were up or down-regulated in both the mms21-1 and mms21-2 backgrounds (Fig 7D). Collectively, the protein/mRNA correlations implied that the lack of MMS21 globally influenced the proteome balance primarily by altering the transcriptome balance.

Role of MMS21 in DNA repair To more specifically address a possible connection between MMS21 and DNA repair [13,43], we compared mRNA abundances for a number of likely contributors as identified by sequence homology to known Arabidopsis factors. From the analysis of this collection by Z-scores, it became apparent that the mms21-1/2 lines had globally altered expression of DNA repair-associated genes, suggesting a dysregulation of the process. For example, of the 66 mRNA analyzed from embryos, many were upregulated in the mutant backgrounds, including those encoding a number of well-described DNA-damage repair factors such as PCNA, NSE1, NSE4a, MRE11b, BRCA1, and multiple subunits of the REPLICATION PROTEIN A (RPA) and RNR complexes, while others were downregulated, including those encoding the DNA mismatch repair protein MSH4 and DMC1 involved in meiotic recombination (Fig 8A and S2 Table). Similar responses were also seen for the few corresponding DNA repair proteins that we could detect by MS, but their measured changes were more muted with most failing to rise above/below a FC = 2 cutoff (p-value<0.05). Only a few showed increases or decreases >2 fold in mms21-2 seedlings, including the DNA mismatch repair protein MLH1, the UVRB/UVC homolog, DRT family members, a Tudor/PWWP ortholog, Whirly1, and SMC5 (Fig 7A). PPT PowerPoint slide

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TIFF original image Download: Fig 8. mms21 Mutants Have an Altered DNA Damage Response. (A) Genes associated with DNA repair have altered expression in the mms21 mutants. Shown are RNA-seq heat maps generated by Z scores from mms21-1 and mms21-2 embryos as compared to those from W22 focused on the altered expression of 66 genes associated with DNA damage repair. Each column represents an individual biological replicate; rows represent specific genes of interest. The numerical Z scores in each box shown standard deviations away from the mean. Groups of genes with similar expression patterns were clustered by columns and rows. (B) mms21 seedlings are hypersensitive to DNA-damaging agents. mms21-1, mms21-2, and W22 seeds were germinated on sterile filter papers and then transplanted to solid growth medium supplemented with 20 ppm methyl methanesulfonate (MMS), 2 μM mitomycin-C (MMC), 1 mM hydroxyurea (HU), 100 nM bleocin (Bleo), or 10 μM zebularine (Zebu). After one-week, root growth, was measured and plotted relative to that seen with untreated W22 roots. Each bar represents the mean of three biological replicates (±SD). (C) Comet assays measuring by electrophoretic mobility the extent of DNA breaks. DNA from nuclei isolated from mms21-1, mms21-2, and W22 roots were subjected to agarose gel electrophoresis and then stained with propidium iodide. Mobility of the DNA was measured by the distance from the center of the nucleus to the edge of the comet tail. Scale bar = 50 μm. (D) Quantification of the comet assays in panel (C) by box plots based on the distribution of comet tail lengths for individual nuclei. The bottom and top of each box indicate the first (Q1) and third (Q3) quartiles, and the middle line reflects the median; the upper-limit equals Q3 plus 1.5 times interquartile range (IQR), and the lower-limit equals Q1 minus 1.5 times IQR. Each dot represents a single measurement (n = 50 cells). https://doi.org/10.1371/journal.pgen.1009830.g008 Observing a potential connection of MMS21 to DNA repair, we next checked the sensitivity of the mms21 mutants to DNA-damaging agents as assayed by the growth of emerging roots. Both mms21-1 and mms21-2 roots were strongly hypersensitive to MMS and mitomycin C, relatively unaffected by hydroxyurea, and slightly affected by bleomycin and zebularine but only for the stronger mms21-2 allele (Fig 8B). The hypersensitivity of mms21 to MMS in particular was consistent with the first discovery of this locus via a yeast MMS-sensitivity screen [44]. And finally, we measured the frequency of DNA strand breaks by comet assays [45]. Here, W22 and mms21 nuclei were isolated from seedling roots, embedded in agarose, and then their DNA was electrophoresed under alkaline conditions; increased DNA breaks were then observed by a greater comet tail length as the DNA migrated toward the anode. As can be seen in Fig 8C and 8D, DNA from the mms21 mutants had more breaks as compared to W22. Taken together with the RNA-seq data, we found that loss of MMS21 impacted DNA integrity and repair, along with substantially altered gene expression that included the ectopic expression of zein-encoding loci. Prior studies with Arabidopsis mms21/hpy2/nse2 mutants implicated MMS21 in endoreduplication, with the mutant shoot nuclei often harboring excess whole genome duplications [12] and pollen having a high frequency of diploid male gametes [29]. When similarly assessed for DNA content by flow cytometry of nuclei, we detected a comparable distribution of 2N and 4N nuclei for 10-DAS mms21-1 leaves as compared to those from wild-type W22, suggesting little impact of MMS21 on cell division in maize somatic tissues (S10A and S10B Fig). Given that endoreduplication is a common feature of maize seeds, especially in the endosperm that undergoes multiple rounds of DNA replication before maturation [46], we then examined the ploidy levels of seed nuclei, using the mms21-2 allele which allowed us to visually discriminate homozygous mutant kernels from their wild-type siblings on the same cob (see S3 Fig). As expected, we detected an expanded series of ploidy levels in whole seeds at 16-DAP, which included 2N, 4N and 8N nuclei in embryos, with the 3N, 6N and 12N nuclei likely representing triploid endosperm nuclei (S10C and S10D Fig). Again, a comparable ploidy distribution was seen for the mms21-2 seed tissues versus W22. Taken together, we were left to conclude that MMS21 has little impact in maize endoreduplication in contrast to that reported in Arabidopsis [12]. However, it is not yet known whether the ploidy levels of male gametes are affected.

MMS21 interacts with SUMO, the SCE1 E2, and the SMC5 and NSE4a Subunits of the SMC5/6 complex We presumed that MMS21 influences DNA dynamics, and ultimately maize development, through SUMOylation of one or more targets. Given: (i) the known connections between MMS21 and the SMC5/6 complex in Arabidopsis and other organisms [13,43], (ii) proteomic indications that Arabidopsis NSE4a is a MMS21 substrate [5], and our discoveries here in maize that (iii) mms21 mutants are hypersensitive to DNA damage, and that (iv) the mRNAs encoding the NSE1 and NSE4a subunits of the SMC5/6 complex are selectively upregulated in mms21 backgrounds, led us to speculate that MMS21 SUMOylates one or more components of the SMC5/6 complex. A further connection was evident when directly quantifying SMC5 protein levels in mms21 seedlings by MS; as shown in Fig 7C, SMC5 levels were significantly lower in the strong mms21 mutants, mms21-2 and mms21-CR1, as compared to their normal siblings. To provide further connections, we examined by yeast two-hybrid (Y2H) assays, whether MMS21 binds maize NSE4a and SMC5 using maize orthologs of the reported interactors BRAHMA and DPa as controls [30,31]. As shown in Fig 9A, MMS21 bound to one of its cognate E2s SCE1b (but not SCE1f) and to DPa, but only poorly to an N-terminal soluble fragment of BRAHMA as judged by growth on selection medium. None of the interactors bound SUMO1a. This association between MMS21 and SCE1b was lost when we used the expected polypeptides derived from the mms21-1 and mms21-2 alleles, implying that these aberrant forms poorly bind the SUMO-E2 intermediate. Intriguingly, MMS21 also bound strongly to SMC5 and NSE4a, with this association only weakly dampened when using the mms21-1 and mms21-2 protein variants (Fig 9A). Because MMS21 and NSE4a likely do not touch each other directly based on a general model of the SMC5/6 complex ([47]; see S14D Fig), we hypothesize that SMC5 helps tether these two proteins. PPT PowerPoint slide

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TIFF original image Download: Fig 9. Maize MMS21 Interacts with the SUMO Conjugation Machinery and Components of the SMC5/6 Complex. (A) Y2H assays testing the interactions between full-length MMS21, or the mms21-1 and mm21-2 truncations, and various components within the SUMO pathway and the NSE4a and SMC5 subunits of the SMC5/6 complex. The known MMS21 interactors, DPa and an N-terminal soluble region of BRAHMA, were included for comparisons. All proteins were derived from maize and were expressed as N-terminal fusions with either the GAL4-activating domain (AD) or DNA-binding domains (BD). BD and AD represent empty vector controls. Shown are colonies grown for 3 d at 28°C on selective medium lacking Leu, Trp, His and adenine (Ade) (top), or on non-selective medium missing only Leu and Trp (bottom). (B) BiFC assays testing pairwise the interactions between several partners shown in panel (A) and wild-type and mutant forms of MMS21. N. benthamiana leaf epidermal cells were co-infiltrated with plasmids expressing the N-and C-terminal fragments of YFP (nYFP and cYFP, respectively) fused to the indicated proteins. Reconstituted YFP fluorescence of epidermal cells along with bright field (BF) views were imaged 40–45 hr after infiltration. Tested pairs were nYFP-MMS21 with cYFP-SUMO1a, nYFP-MMS21 with cYFP-SCE1b or cYFP-SCE1f, nYFP-MMS21 with cYFP-NSE4a, and nYFP-SMC5 with cYFP-MMS21. Additional BiFC control images are found in S11 Fig. Note that MMS21 interacts in both the cytoplasm and nucleus with the SUMOylation machinery but only in the nucleus with NSE4a and SMC5. Nuc, nucleus. Scale bars = 20 μm. https://doi.org/10.1371/journal.pgen.1009830.g009 To further validate the MMS21 interactions in planta, we applied bimolecular fluorescence complementation (BiFC) assays that transiently expressed maize SUMO1a, SCE1b, SCE1f, SMC5 and NSE4a as fusions with the N-terminal and C-terminal fragments of YFP in Nicotiana benthamiana epidermal cells: interactions were then scored by reconstituted YFP fluorescence. Even though we failed to detect interactions between MMS21 and SUMO1a by Y2H, strong BiFC signals was evident in the nucleus and cytoplasm of leaf cells co-expressing MMS21 and SUMO1a (Figs 9B and S11), confirming the expectation that MMS21 interacts with SUMO1a in planta. Likewise, we detected BiFC interactions between MMS21 and SCE1b and now weak interaction of MMS21 with SCE1f in both the nucleus and cytoplasm. Most interestingly, N. benthamiana cells co-expressing MMS21 with NSE4a or SMC5 also reconstituted YFP fluorescence but these signals were only evident in the nucleus, consistent with the known nuclear location of the SMC5/6 complex (Fig 9B). While the mms21-1 protein appeared to retain its affinity for NSE4a and SMC5 based on the BiFC signals, this affinity appeared less strong for the mms21-2 protein, potentially in agreement with its more compromised architecture.

[END]

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